This invention relates to the identification and use of cis acting nucleic acid elements that bind to nucleic acid binding factors to regulate genetic activities of nucleic acids.
All living creatures store information in nucleic acid molecules called DNA or RNA that encode structural and regulatory proteins. The collective behavior of nucleic acids and proteins constitutes and controls normal cell and organismal life cycles. Nucleic acids and proteins also act as causative agents in, or response factors to, pathological conditions.
Transcription of DNA into RNA, translation of RNA into proteins and other genetic events such as nucleic acid synthesis, sorting, processing, repair and degradation, are regulated by a variety of specialized nucleic acid binding factors. Nucleic acid binding factors bind to specific sequences present on the nucleic acid molecules they regulate, called cis acting nucleic acid elements. These nucleic acid binding factors, bound to their specific cis acting nucleic acid elements, are able to interact with other cellular factors to modulate specific genetic events. The binding of a nucleic acid binding factor to a cis acting nucleic acid element, or its ability to interact with other factors that mediate genetic events, or both, can be regulated in response to signals transmitted into the cell from the cell exterior.
As an example, regulatory proteins called "transcription factors" bind to cis acting nucleic acid elements on genomic DNA at sites known as "promoters" and "enhancers" present at variable distances from the site of initiation of transcription of the genes they regulate. The enhancer sequences and adjacent nucleic acid sequences, together with their bound transcription factors, are able to bend to contact the transcriptional complex bound to the promoter. Such contact can either enhance or reduce expression of the regulated gene.
The human genome, which stores the genetic information of a human cell as DNA, is estimated to contain about 100,000 genes. Each of these genes and the RNAs they encode is likely to have multiple cis acting nucleic acid elements that bind to corresponding nucleic acid binding factors to regulate gene expression. These cis acting nucleic acid elements, and the factors that bind them, are potential targets for therapeutic drugs that could be used to modulate gene expression. Determining which cis acting nucleic acid elements are bound under different conditions can also be used to characterize and monitor the genetic responses of a cell under normal, pathological or experimental conditions.
Current methods of identifying cis acting nucleic acid elements have several disadvantages. Most of these methods require prior identification of either the nucleic acid that is regulated, or the corresponding regulatory nucleic acid binding factor, or both. For example, once a nucleic acid has been identified, adjacent sequences, which are predicted to contain cis acting nucleic acid elements, can be isolated and subsequences therefrom are tested for cis activities. Alternatively, once a nucleic acid binding factor has been isolated, the sequences to which it binds can be identified. Other methods, which are limited to identifying transcriptional enhancer elements, involve cloning random nucleic acid sequences upstream of a reporter gene and observing expression of the reporter gene product.
At present, however, there is no broadly applicable method to identify cis acting nucleic acid elements without prior identification of the regulated nucleic acid or of the regulatory nucleic acid binding factor. There is also no rapid and efficient method to simultaneously identify a plurality of cis acting nucleic acid elements.
Thus, there exists a need for a method of rapidly and efficiently identifying cis acting nucleic acid elements. The present invention satisfies this need and provides related advantages as well.